SET, also known as protein phosphatase 2A inhibitor 2 protein (I2PP2A), putative HLA-DR associated protein II (PHAPII), inhibitor of granzyme A-activated protein II (IGAAD) and template-activating factor (TAF1β), was first described as part of the SET-CAN fusion gene in a patient with acute undifferentiated leukemia, apparently as a result of a gene translocation (Von Lindern et al., 1992, Mol. Cell. Biol. 12: 3346-3355). SET has since been characterized as a multifunctional protein that protects histones from acetylation by histone acetyl transferases, modulates HuR mRNA binding, regulates G2/M transition via binding to p21CIP1, and acts as a transcription factor for P450c17 activation (Seo et al., 2001, Cell. 104: 119-130; Brennan et al., 2000, J. Cell Biol. 151: 1-14; Canela et al., 2003, J. Biol. Chem. 278: 1158-1164; Compagnone et al., 2000, Mol. Endocrinol. 14: 875-888).
More recently, it has been suggested that SET plays a role in Alzheimer's disease (AD) and other neurodegenerative diseases (Madeira et al., 2005, FASEB J. 19: 1905-1907; Tsujio et al., 2005, FEBS Letters. 579: 363-372). Evidence of such SET activity includes a finding of increased SET expression in the hippocampus of AD patients which correlates positively with neurofibrillary tangles and negatively with Mini-Mental Status Exam (Blalock et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101: 2173-2178). SET has also been found to play an important role in the regulation of cell death induced by a pro-apoptotic domain of the amyloid precursor protein (APP), a protein seen in the brains of AD patients (Madeira et al., 2005, FASEB J. 19: 1905-1907). When overexpressed, the short cytoplasmic domain of APP referred to as “Jcasp” activates caspase-3 and induces neuronal death. SET specifically binds to Jcasp, and down-regulation of SET reduces Jcasp-induced cell death. Conversely, SET gain of function increases cell death.
Although much still needs to be done to elucidate the role of SET in neurological diseases, SET has been found to be a potent inhibitor of protein phosphatase 2A (PP2A), a phosphatase involved in the regulation of diverse cellular processes (Li et al., 1996, J. Biol. Chem. 271: 11,059-11,062). The inhibition of PP2A by SET appears to be substrate specific. It has been demonstrated that SET inhibits PP2A when using phosphorylated myelin basic protein as a substrate but does not inhibit the activity of PP2A when using phosphorylated casein as the substrate (Guo et al. 1995, Biochemistry, 34, 1988). PP2A appears to be deactivated by phosphorylation and activated by methylation of its C subunit (Chen et al., 1992, Science. 257, 1261-1264; Guo and Damuni, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 2500-2504; Favre et al., 1994, J. Biol. Chem. 269: 16311-16317).
PP2A dephosphorylates tau and MAP2 in vitro (Yamamoto et al., 1988, J. Neurochem. 50: 1614-1623). Tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play an important role in the assembly of tubulin monomers into microtubules and stabilize neuronal microtubule networks. Microtubules are involved in maintaining the cell shape and serve as tracks for axonal transport. Tau proteins also establish some links between microtubules and other cytoskeletal elements or proteins. Their expression is developmentally regulated by an alternative splicing mechanism, and six different isoforms exist in the human adult brain. Further, tau proteins are the major constituents of intraneuronal and glial fibrillar lesions described in Alzheimer's disease and numerous neurodegenerative disorders referred to as tauopathies. Molecular analysis has revealed that an abnormal phosphorylation of tau might be one of the important events in the process leading to their detachment from microtubules, aggregation into filamentous structures and/or stabilization of filamentous structures comprising paired helical filaments and/or neurofibrillary tangles (Buee et al., 2000, Brain Res. Rev. 33: 95-130).
The activities of PP2A are believed to be compromised in Alzheimer's disease brain. In AD brain, it has been speculated that PP2A deficiency permits hyperphosphorylation of tau leading to neurofibrillary tangle formation and neuronal degeneration. Evidence supporting this hypothesis includes the finding that in vitro treatment of metabolically active rat brain slices with okadaic acid causes inhibition of PP2A activity and abnormal hyperphosphorylation of tau resulting in the inability of tau to bind to microtubules (Gong et al., 1995, J. Neurochem. 65: 732-738). As described above, SET is overexpressed in the hippocampus of Alzheimer's patients relative to normal people. The observed in vivo overexpression would result in inhibition of PP2A, in a manner similar to okadaic acid, and would be expected to lead to hyperphosphorylated tau and neurofibrillary tangle formation. It has also been reported that phosphorylation at a site, which is recognized by PP2A, is required for trafficking of β-secretase to the surface of cells where it is active in cleavage of the amyloid precursor protein (APP) (Walter et al. 2001, J Biol Chem. 276, 14634). Cleavage of APP by β-secretase initiates a proteolytic pathway that results in the production of amyloid-β (Aβ) protein. Further, as described above, methylation of PP2A is required for full activity. Treatment with S-adenosylhomocysteine leads to decreased methylation of PP2A, reduces PP2A activity, and has been shown to result in increased production of Aβ due to increases in phosphorylation of the soluble APP that leads to β cleavage (Sontag et al., 2007, J. Neurosci. 27: 2751-2759). Therefore, activation of PP2A may reduce the level of Aβ in the brains of Alzheimer's patients by preventing the trafficking of the secretase to the surface or by decreasing the phosphorylation that targets APP towards β cleavage that is required for Aβ formation.
PP2A has also been shown to interact with, and dephosphorylate a number of proteins involved in the signal transduction cascades that propagate inflammatory signaling processes. One such protein is the Inhibitor of NFκB kinase (IκK). IκK is activated by phosphorylation, and in turn phosphorylates the Inhibitor of NFκB (IκB) (Hong et al. 2007 J. Biol. Chem.) Phosphorylation of IκB results in release of Nuclear Factor κB (NFκB) from the inactive IκB:NFκB complex and the degradation of IκB. This leaves NFκB free to become phosphorylated and translocated to the nucleus where it acts as a transcription factor that controls gene expression of pro-inflammatory cytokines Activation of NFκB has also been shown to be required in the process of antigen presentation (Yoshimura et al. Scan. J. Immunol. 58, 165), a process that is integral to innate immunity and autoimmune disorders.
PP2A has also been reported to bind to and dephosphorylate the p38 mitogen activated protein kinase (MAPK), thus inactivating it (Sundaresan & Farndale 2002 FEBS Letters 528, 139). Activation of p38 and other MAPKs is required for production of pro-inflammatory cytokines and T-cell proliferation. Another protein implicated in inflammatory signaling that may be dephosphorylated by PP2A is IL-1beta receptor-associated kinase (IRAK). IRAK is integral in interleukin signaling and signaling cascades stemming from the Toll-like receptor family proteins.
Apolipoprotein E (ApoE) is another protein that has been shown to play an important role in neurological disease and has immunomodulatory properties. ApoE has been demonstrated to have immunomodulatory effects in vitro, including suppression of lymphocyte proliferation and immunoglobulin synthesis after mitogenic challenge. ApoE is secreted in large quantities by macrophages after peripheral nerve injury, and by microglia, astrocytes and oligodendrocytes (glial cells) after CNS injury.
Human ApoE is found in three major isoforms: ApoE2, ApoE3, and ApoE4; these isoforms differ by amino acid substitutions at positions 112 and 158. The most common isoform is ApoE3, which contains cysteine at residue 112 and arginine at residue 158; ApoE2 is the least common isoform and contains cysteine at residues 112 and 158; ApoE4 contains arginine at residues 112 and 158. Additional rare sequence mutations of human ApoE are known (see, e.g., Weisgraber, 1994, Advances in Protein Chemistry 45:249, 268-269).
It has been observed that ApoE influences development of late onset and familial AD. This effect is robust and dose-dependent, such that homozygous individuals with an APOE4/4 genotype have an approximately 20-fold increased risk of developing AD, and heterozygous individuals with an APOE3/4 genotype have a 4-fold increased risk relative to patients who are homozygous for the most common APOE3/3 genotype (Strittmatter et al., 1993; Corder et al., 1993; reviewed by Laskowitz et al., 1998a). This observation has led to a resurgence of interest in the function of ApoE in the mammalian central nervous system (CNS). Because of its association with AD, multiple laboratories have examined interactions between ApoE and proteins believed to play a role specific to the pathogenesis of AD. Further, several laboratories have described isoform-specific interactions between ApoE and Abeta or ApoE and tau (Strittmatter et al. 1994; Gallo et al. 1994; Fleming et al. 1996; reviewed by Laskowitz et al., 1998a). The role of ApoE in the CNS, however, remains undefined, and it is unclear which or any of these interactions are relevant in human neurodegenerative disease.
It has previously been found that certain ApoE peptide derivatives are useful for treating inflammation and neurological disorders including traumatic brain injury (TBI). In this regard, U.S. application Ser. No. 10/252,120, filed Sep. 23, 2002 (herein incorporated by reference in its entirety), discloses methods of using ApoE analogs, including COG 133, to treat or ameliorate the neurological effects of cerebral ischemia or cerebral inflammation. COG 133 is a small peptide, comprised of residues 133-149 of the ApoE protein. U.S. Application No. 60/606,506, filed Sep. 2, 2004, and U.S. Application No. 60/608,148, filed Sep. 9, 2004 (herein incorporated by reference in their entireties), disclose the use of COG 133, COG 1410 and other ApoE derivatives to treat traumatic brain injury and diseases involving inflammation. COG 1410 is a mutated derivative of COG 133 that exhibits a 4-fold gain in therapeutic window and a 7.4-fold gain in Therapeutic Index as compared to COG 133.
Despite recent efforts in elucidating the role of SET in neurological disease, there has not previously been an identified connection between SET and ApoE. The present inventors have surprisingly found that COG 133 and other ApoE derivative peptides bind to and modulate the activity of the SET protein. The present invention thus provides a novel method of modulating PP2A activity by blocking SET binding to PP2A using exogenous agents such as ApoE derivatives. The present invention also provides methods of modulating other activities of SET, including enhancement of Cdk5 activity and increased Jcasp-induced neuronal apoptosis, by interfering with SET binding to those respective targets. Such novel methods can be used for the treatment of neurological, inflammatory, and other diseases as well as for screening drug candidates for efficacy in the treatment of neurological, inflammatory, and other diseases.